Porphyromonas gingivalis-odontogenic infection is the potential risk for progression of nonalcoholic steatohepatitis-related neoplastic nodule formation

Porphyromonas gingivalis (P.g.), a major periodontal pathogen is a known risk factor for various systemic diseases. However, the relationship between P.g. and nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is unclear. Thus, we aimed to elucidate whether P.g.-odontogenic infection promotes NASH-related HCC development/progression and to clarify its mechanism. Using high-fat diet (HFD)-induced NASH mouse model, P.g. was infected odontogenically. After 60 weeks of infection, tumor profiles were examined. Chow diet (CD) groups were also prepared at 60 weeks. Nodule formation was only seen in HFD-mice. P.g.-odontogenic infection significantly increased the mean nodule area (P = 0.0188) and tended to promote histological progression score after 60 weeks (P = 0.0956). Interestingly, P.g. was detected in the liver. HFD-P.g. (+) showed numerous TNF-α positive hepatic crown-like structures and 8-OHdG expression in the non-neoplastic liver. In P.g.-infected hepatocytes, phosphorylation of integrin β1 signaling molecules (FAK/ERK/AKT) was upregulated in vitro. In fact, total AKT in the liver of HFD-P.g. (+) was higher than that of HFD-P.g. (−). P.g.-infected hepatocytes showed increased cell proliferation and migration, and decreased doxorubicin-mediated apoptosis. Integrin β1 knockdown inhibited these phenotypic changes. P.g.-odontogenic infection may promote the progression of neoplastic nodule formation in an HFD-induced NASH mouse model via integrin signaling and TNF-α induced oxidative DNA damage.


Results
Tumor profiles in HFD-induced NASH mouse model due to P.g.-odontogenic infection. At the 60 weeks after P.g.-odontogenic infection, the body and liver weight of the HFD-60w-P.g. (+) and HFD-60w-P.g. (−) groups were significantly increased compared with those of the CD-60w-groups ( Supplementary Fig. 1a,b). In CD and HFD groups, there was no change in body and liver weight between mice with and without P.g.odontogenic infection ( Supplementary Fig. 1a,b).
The alveolar bones of the first molar root apices of all CD/HFD-P.g. (+) groups were markedly destroyed, and periapical granulomas were formed (Fig. 1a). No significant difference was found in the condition of periapical granulomas between the CD-P.g. (+) and HFD-P.g. (+) groups. Immunohistochemically, numerous P.g.-positive granules were found in the infected pulp and periapical granulomas (Fig. 1b). Figure 1c shows a high-power view of the periapical granuloma. Numerous P.g. were phagocytosed by Mφ (arrowheads), but some P.g. (arrows) were diffusely distributed among the cells. Figure 1d shows the macroscopic findings of the liver. In both HFD-60w-P.g. (+) and (−) groups, the livers were large and yellowish because of fatty deposition. Meanwhile, the CD-60w groups generally had small and reddish-brown livers.
Histologically, liver carcinogenesis is a multistep process 14 . In mouse HCC, it sequentially develops from precancerous lesions, including the focus of cellular alteration (early stage of hepatocarcinogenesis) and hepatocellular adenoma (late stage) ( Supplementary Fig. 2b) 15 .
Changes in macrophages (Mφ), hepatic crown-like structures (hCLSs), and TNF-α expression in the non-neoplastic liver due to P.g.-odontogenic infection. hCLSs, which is composed of Mφ surrounding the degenerated hepatocytes (an indicator of NASH inflammation and fibrosis), is accepted to correlate not only the onset of chronic inflammation and fibrosis 17 but also HCC development in NASH 18 . hCLSs can produce TNF-α, which plays a critical role in hepatocarcinogenesis 19 . Therefore, we considered that increased hCLSs with P.g.-infection might be associated with progression of nodule formation through TNF-α production. Immunohistochemically, the number of Mac2-positive Mφ in the non-neoplastic liver of the HFD-60w-P.g. (+) group was significantly higher than that of HFD-60w-P.g. (−) group (Fig. 3a,b; P = 0.0135). Furthermore, the number of hCLSs in the HFD-60w-P.g. (+) group was also significantly higher than that of HFD-60w-P.g.  Figure 3d showed Immunohistochemistry of TNF-α in non-neoplastic liver. In the HFD-60w-P.g. (+) group, the hCLSs showing intensive TNF-α positivity were more abundantly scattered than in the HFD-60w-P.g. (−) group. Hepatocytes were weakly positive for TNF-α. The number of TNF-α-positive hCLSs in the HFD-60w-P.g. (+) group was significantly higher than that in HFD-60w-P.g. (−) group (P = 0.001; Fig. 3e). Moreover, IL-6 was checked because it is also known tumor promoting cytokines for NASH-related HCC 19 . IL-6 was significantly upregulated in HFD-60w-P.g. (+) group (P = 0.0408; Supplementary Fig. 3a,b). In our study, P.g.-induced exacerbation of NASH was significantly severe. NASH progression is said to be closely related to oxidative stress 20 . Therefore, we considered that oxidative stress caused by hCLSs-induced TNF-α may produce 8-OHdG to cause nodule formation.
Changes in the oxidative DNA damage in the non-neoplastic liver due to P.g.-odontogenic infection. TNF-α is capable of causing oxidative DNA damage 21 . It has been reported that oxidative DNA damage triggered by various factors such as inflammation promotes hepatocarcinogenesis, and 8-hydroxy-2-deoxyguanosine (8-OHdG), oxidative DNA damage marker which can cause G: C to T: A mutation, is useful for predicting NASH-related HCC development 20,22 . As expected, 8-OHdG was significantly increased in HFD-60w-P.g. group (P = 0.0029; Fig. 4a,b). It was stained with both nuclei and cytoplasm. These results indicate that hCLSs-induced TNF-α upregulation caused by P.g.-odontogenic infection promote neoplastic nodule formation through mutagenic 8-OHdG in an HFD-induced NASH mouse model. Changes in the integrin-FAK pathway in hepatocytes due to P.g.-infection. Immunohistochemically, P.g. was mainly detected in hepatocytes in non-neoplastic liver of P.g. (+) groups. Interestingly, P.g. can bind to integrin α5β1 in oral epithelial cells leading to integrin signaling activation 23 . Furthermore, Integrin β1 signaling is involved in HCC development/progression 24,25 and drug resistance in HCC 25 . Therefore, in this study, we focused on integrin β1-FAK signaling in nodule formation.

Discussion
In this study, P.g.-odontogenic infection induced progression of neoplastic nodule formation in the liver of an HFD-induced NASH mouse model. To the best of our knowledge, this is the first study to confirm the role of P.g.-odontogenic infection in NASH-related neoplastic nodule formation.
In an HFD-induced NASH mouse model, NASH was induced only by HFD without the addition of chemicals or genetic modifications. As such, we consider that a pathological condition mimicking human NASH could be created. It has been reported that liver neoplastic nodule formation occurred in approximately half of C57B1/6J mice after 60 weeks of HFD feeding 26 . We also found neoplastic nodules in the liver of 66.7% (8/12 mice) of the mice in the HFD-60w-P.g. (−) group, indicating that it is appropriate as a research model to examine the The ratio of phosphorylated protein/total protein was calculated and compared using ImageJ software, ver.1.52a (https:// imagej. net/ ij/ docs/ index. html). Significant overexpression of total AKT is confirmed (*P < 0.05, Mann-Whitney test). β-actin is used as an internal control for phosphorylated AKT and total AKT. CD-P.g chow diet Porphyromonas gingivalis group, HFD-P.g high-fat diet Porphyromonas gingivalis group, hCLSs hepatic crownlike structures, TNF tumor necrosis factor.  www.nature.com/scientificreports/ effects of odontogenic infection of periodontal pathogens on NASH-related atypical/neoplastic nodule formation including HCC. P.g. is a main periodontal pathogen that requires anaerobic conditions for growth. It is well accepted that P.g. is detected not only in deep periodontal pocket, but also in distant organ like arteriosclerosis plaque or brain suffering to Alzheimer disease, resulting in occurrence and/or exacerbation of systemic diseases. Moreover, P.g. was immunohistochemically detected in the tumor area of esophageal cancer and was associated with cancer differentiation, lymph node metastasis, and TNM stage 12 . It is reported that P.g. is detected in infected pulp chamber with periapical periodontitis as a second major bacterium next to P. endodontalis and associated with moderate to high cardiovascular risk 27 . Pereira et al. confirmed that P.g. is more prevalently detected together with P. intermedia in pulp chamber of endodontic-periodontal disease 28 . Therefore, in the present study, P.g. was selected as infecting bacterium, and to obtain anaerobic conditions suitable for P.g. growth, we applied P.g. from pulp chamber, which ideally provided the natural infection way of periapical periodontitis. Previously we confirmed that P.g. was alive inside the pulp chamber and reproduced itself over a long period to induce periapical periodontitis and exacerbated inflammation and fibrosis of NASH 9,10 . In the present study, after 60 weeks of P.g.-odontogenic infection, numerous P.g. were detected in the pulp and periapical granuloma and were distributed in the liver. The average NAS in the liver of HFD-60w-P.g. (+) group was significantly higher than that in HFD-60w-P.g. (−) group, indicating that periapical granuloma still functioned as reservoir of P.g. to aggravate NASH progression.
Interestingly, in CD mice with or without P.g.-odontogenic infection not only HCC but also tumor nodule formation indicating precancerous lesion was not observed.
Using the HFD-induced NASH mouse model with short period of P.g.-odontogenic infection (6 weeks), we previously confirmed that P.g.-odontogenic infection exacerbated inflammation/fibrosis of NASH, in which mechanism TLR2 upregulating and/or inflammasome activating by lipid accumulation in hepatocytes contributed to excessive production of proinflammatory cytokines.
In other words, fatty liver has an increased susceptibility to P.g.-LPS/lipoprotein. Therefore, it is suggested that P.g.-odontogenic infection like periodontitis can more aggressively exacerbate NASH-related neoplastic nodule formation in obese individual than normal individuals. Actually, the mean nodule area of the HFD-60w-P.g. (+) was significantly larger than that of HFD-60w-P.g. (−) group (P = 0.0188), indicating P.g.-odontogenic infection accelerated neoplastic nodule formation including precancerous and HCC nodules. Unfortunately, we could not get statistical difference in HCC development rate. In fact, 4 out of 12 mice (33.3%) progressed to HCC in HFD-60w-P.g. (+) group while only one out of 12 mice (8.3%) had HCC in the HFD-60w-P.g. (−) group. Considering all of data together, it is indicated that P.g.-odontogenic infection is an important risk factor for neoplastic nodule formation in the NASH liver of HFD-induced NASH mouse model.
It is well accepted that proinflammatory cytokines like TNF-α also correlated with development and/or progression of inflammation related cancers such as HCC. We considered that proinflammatory cytokines produced in the NASH liver with P.g. infection is potential risk for nodule formation.
In this study, TNF-α positive hCLSs were significantly increased in the non-neoplastic liver in the HFD-60w-P.g. (+) group. TNF-α is associated with HCC, colitis-associated cancer, and gallbladder cancer. In the liver, TNF-α induces reactive oxygen species (ROS). ROS causes DNA damage, which initiates tumorigenesis 29 . Autocrine TNF-α was reported to act as a tumor promoter gene by promoting gallbladder cancer cell proliferation via the AKT/NF-κB/Bcl-2 pathway 30 . In mouse hepatocytes, TNF-α phosphorylated AKT, which plays an important role in the regulation of cell proliferation and apoptosis 31 . Moreover, ERK is another downstream molecule of TNF-α signaling pathway. ERK expression was attenuated in HCC progenitor cells isolated from DEN-treated TNFR1-knockout mice, in which HCC development was suppressed 32 . TNF-α is also known to promote FAK activation in epithelial cells, endothelial cells, fibroblasts and muscle cells to cause various cellular responses such as inflammatory cytokine, matrix metalloproteinase, or VCAM-1 production 33 . Especially, Corredor et al. have reported that TNF induces intestinal epithelial cells migration via TNFR2 initiated FAK phosphorylation 33,34 . Furthermore, Pokharel et al. mentioned that TNF-α promotes the production of 25-hydroxycholesterol (25HC), a lipid ligand for integrins, and induces pro-inflammatory response (CCL3 production) via integrin α5β1-FAK signaling 33 . Our in vitro experiments confirmed that P.g. directly activates integrin β1 signaling. Moreover, in vivo data also showed a tendency to activate FAK, AKT, and ERK. These data indicate that not only P.g. but also TNF-α may promote hepatocyte proliferation, anti-apoptosis and migration via integrin signaling. Nakagawa et al. hypothesized that NASH-HCC development was dependent on TNF-α produced by Mφ. The main source of TNF-α is hCLS, which is positively correlated with chronic inflammation and liver fibrosis 17 . In our study, IHC of TNF-α showed an increase in the number of TNF-α-positive hCLS in the HFD-60w-P.g. (+) group, indicating that TNF-α produced from Mφ and hCLS stimulated with P.g. may play an important role in progression of NASH-related nodule formation. TNF-α induces reactive oxygen species (ROS). ROS causes DNA damage which initiates tumorigenesis 29 . Maki et al. considered that DNA damage caused by HCV-induced chronic inflammation is associated with HCV-associated HCC development because 8-OHdG and CD68, Mφ marker, of noncancerous liver tissues of HCV-associated HCC patients was significantly upregulated compared to those of metastatic liver cancer patients, and significant positive correlations were confirmed between CD68-positive inflammatory cells and 8-OHdG in liver tissues of HCV-associated HCC patients 35 . Tanaka et al. performed immunostaining of 8-OHdG in cancerous and non-cancerous liver tissues of NASH-related HCC patients, and found that 8-OHdG was significantly increased compared to HBV-associated HCC and ALD-associated HCC patients suggesting that NASH-related HCC development is highly associated with oxidative DNA damage via ROS production 20 . It was also confirmed that 8-OHdG in NASH-related HCC patient liver tissue increased significantly compared to simple fatty liver patients and NASH patients 36 . They speculated that ROS production not only promotes inflammation but also promotes hepatocarcinogenesis, and concluded that 8-OHdG is useful for predicting NASH-related HCC development 20  www.nature.com/scientificreports/ HFD-60w-P.g. (+) group. This implies that increased TNF-α-positive hCLSs in the liver of HFD-P.g. (+) group promote NASH-related nodule formation through the oxidative DNA damage.
Integrins are receptors of various extracellular matrices and consist of α and β subunits 37 . P.g. binds to integrin α5β1 in oral epithelial cells and leads to integrin signaling activation and cell invasion 23 . Interestingly, Helicobacter pylori binds to integrin α5β1 and is involved in stomach cancer development by activating FAK 38 . Integrin β1 signaling has been reported to be involved in HCC development/progression 24,25 . Moreover, drug resistance in HCC is correlated with integrin β1-FAK/AKT signaling 25 . Therefore, we focused on the integrin β1 pathway as another possible mechanism. We found that phosphorylations of FAK, AKT, and ERK were enhanced in P.g.infected cells, in which cell proliferation and migration were upregulated, whereas apoptosis was downregulated. Integrin β1 knockdown significantly reduced the proliferation of P.g.-infected cells, probably because integrin β1 signaling was suppressed and only P.g.-induced damage to hepatocytes remained. P.g.-infection also induces anti-apoptotic effects and activates cell proliferation by activating the AKT signaling pathway in gingival epithelial cells 39 . In particular, AKT plays a central role in various signal networks and is attracting attention as a therapeutic target for HCC 40 . Surprisingly, the total AKT in the NASH liver of the HFD-60w-P.g. (+) group was significantly upregulated. It is reported that AKT1 expression is higher in HCC cells than in normal hepatocytes, and AKT1 suppresses the phosphatase and tensin homolog and upregulates Notch1 to cause HCC growth and anti-apoptosis 41 . However, the mechanism by which P.g.-infection induces the upregulation of the total AKT in the NASH liver remains unclear. This should be clarified in the future.
This study has potential limitations. Since in this experiment, samples size are fairly small and/or observation period is too short, we could not proof the significant difference in HCC development rate between HFD-P.g.

Conclusion
Our data suggests that P.g.-odontogenic infection might accelerate NASH-related neoplastic nodule formation by activating cell proliferation, anti-apoptosis, and cell migration via the integrin β1-signaling pathway such as FAK, AKT and ERK, and by oxidative DNA damage caused by TNF-α produced from increased Mφ that formed hCLS (Fig. 7).

Methods
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Periodontal tissues and livers were evaluated at 60 weeks after P.g.-odontogenic infection immunohistochemically and histomorphometrically.
This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Hiroshima University Animal Research Committee and AVMA Guidelines on Euthanasia. This experimental design was approved by the Hiroshima University Animal Experiment Ethics Committee (permit no. # A17-2). This study complied with ARRIVE 2.0 guidelines for preclinical animal study. All mice were housed in a specific pathogen-free facility in 12-h light-dark cycles with access to water and food ad libitum, and health monitoring was conducted daily. All mice were male. Mice were divided into four groups at random. Up to five mice were kept in one cage (1-3 mice: small cage, 4, 5 mice: large cage).
In order to exclude the possibility of the carcinogenic effects on hepatocarcinogenesis induced by chronic inflammation of the injured area.
One CD-60w-P.g. (−), one CD-60w-P.g. (+), four HFD-60w-P.g. (−) and one HFD-60w-P.g. (+) mice were excluded because they were injured due to fighting or diffuse ventral decubital ulcer caused by cage in too much obese. Optical density (OD value) was measured at a wavelength of 660 nm using a spectrophotometer: SPEC-TRONIC 200 (Thermo Fisher SCIENTIFIC) to measure the cell concentration of P.g. diluted with PBS.
Macroscopic analysis of liver nodules. After careful observation of the liver surface, 2-mm sections of each liver lobe were continuously resected. The cut surface was evaluated if there were 1-mm nodules that could be investigated macroscopically. The diameter (mm) of the largest nodule in each mouse was measured, and was categorized as no tumor, < 4 mm or ≥ 4 mm. The area of nodules was calculated and compared using polygon selections of ImageJ software, ver.1.52a (https:// imagej. net/ ij/ docs/ index. html). The number of nodules in each mouse was also counted, and was categorized as no tumor, 1, ≥ 2. Nodules were histologically divided into precancerous lesions (score 1) or HCC (score 2). Mice with no nodules were scored as 0 in each nodule profile category. Nodules of HFD-48w-P.g. (−) and P.g. (+) groups were evaluated with the macroscopic findings of the liver surface because all but one mouse had no nodules.
Histopathological analysis. Liver tissues were immersed in PLP fixative for 48 h and immediately embedded in paraffin according to routine methods.  (1) integrin signaling, i.e., P.g. activates integrin signaling including FAK, AKT, and ERK, which promotes proliferation, antiapoptosis, and migration, and (2) TNF-α produced from hCLSs may also induce activation FAK, AKT and ERK, which are downstream of integrin-signaling, and accelerate proliferation, anti-apoptosis and migration of P.g.infected hepatocytes. TNF-α production leads to oxidative DNA damage and mutagenic 8-OHdG production involved in nodule formation. The two mechanisms finally result in nodule formation. TNF-α tumor necrosis factor-α. www.nature.com/scientificreports/ Periodontal tissues were demineralized using 10% EDTA-4Na phosphate buffer solution (Sigma-Aldrich Japan Co., Tokyo, Japan) for 4 weeks after PLP fixation. Then 4.5 μm thickness of paraffin sections were stained with HE. The histopathological diagnosis of the nodules and evaluation of the underlying NASH liver were performed by two pathologists. NAS was calculated based on Kleiner's criteria (Supplementary Table 1) by scoring steatoses (0-3), lobular inflammation (0-3), and hepatocyte ballooning (0-2) in four pictures at × 200 magnification 16 . Changes in periapical tissue were examined using hematoxylin and eosin-stained specimens.
Western blotting. Tissue lysate was collected from non-neoplastic liver tissue of each animal. β-Actin, FAK, ERK and AKT protein expression was detected according to the following method. Mice liver tissue were cut into 2 × 2 mm in size and mixed with Tissue Extraction Reagent (Thermo Fisher SCIENTIFIC) including Proteinase inhibitor cocktails (Sigma-Aldrich Japan Co.) and homogenized. After centrifugation of the lysate at 10,000 rpm at 4 °C for 5 min, the supernatant was collected, and the protein concentration was quantitated with a XL-Bradford (integrale, Tokyo, Japan).
Thereafter, the sample was dissolved in Laemmli buffer and kept at 100 °C for 3 min and separated by polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane (Schleicher & Schuell, Dasse, Germany). Blocking was carried out for 1 h at room temperature using 5% skim milk and the primary antibody was acted and overnight at 4 °C. The following antibodies were used as primary antibodies.
The secondary antibody was applied at room temperature for 1 h. Anti-mouse IgG, HRP-linked antibody (# 7076, Cell Signaling Technology Japan, KK) or Anti-rabbit IgG, HRP-linked antibody (# 7074, Cell Signaling Technology Japan, KK) was used as a secondary antibody. Bands were visualized by chemiluminescence using ECL western blotting detection system (Western Lightning ECL Pro), (PerkinElmer, INC., Massachusetts, USA). The blots were cut prior to hybridization with antibodies, and cropped images are put in the figures. We put uncropped images of blots with membrane edges visible in Supplemental Information file. Laboratory investigations. Cell culture. Immortalized human fetal hepatocytes (HC3716-hTERT), kindly provided by Prof. Hidetoshi Tahara (Hiroshima University), were cultured in Hepatocyte Culture Media SingleQuots (Lonza, Walkersville, USA) added with Hepatocyte Basal Medium (Lonza) and supplemented with heat-inactivated 15% fetal bovine serum (Cytiva, Tokyo, Japan).
Detection of integrin-β1-signaling pathway molecules by Western blotting. HC3716-hTERT cells were infected with P.g. at multiplicity of infection (MOI) of 100. After 15, 30, and 45 min of P.g.-infection, protein was collected. Phosphorylations of FAK, AKT, and ERK were detected by Western blotting.
Proliferation assay. HC3716-hTERT cells infected with P.g. at MOI of 100 were seeded in 24-well plates at a density of 1 × 10 4 cells/well. The number of cells on days 1 and 5 of infection was counted with the Beckman Coulter Z1 Particle Counter (BECKMAN COULTER Life Sciences, Indianapolis, USA), and the growth rate was determined.
Assessment of apoptosis by Western blotting. One day after P.g.-infection of HC3716-hTERT cells at MOI of 100, Dox was applied for 18 h, and proteins were collected. Apoptosis markers (cleaved caspase-3 and poly(ADPribose)polymerase [PARP]) were examined by Western blotting.
Wound healing assay. 9 × 10 5 HC3716-hTERT cells were seeded on a 6-well plate. After 24 h, the cells were infected with P.g. at MOI 100 for 2 h. Then, they were washed with phosphate-buffered saline, and the wound area was scratched with a pipette tip. The wound areas were measured at 0 h and 24 h, and the closure areas were calculated using an inverted phase-contract microscope (NIKON INSTECH CO., LTD. Tokyo, Japan; 4× objective). The distances of the 4 closest points in one wound were measured and set to n = 4.
Statistical analysis. Results are reported as the mean ± standard deviation (SD). Unpaired t-test, Fisher's exact test, Mann-Whitney test or Kruskal-Wallis test followed by Dunn's post hoc test were used to analyze the data with the level of significance set at P < 0.05. Statistical between-group differences were evaluated using analysis of variance with the level of significance set at P < 0.001, P < 0.01 and P < 0.05. All statistical analyses were performed using R (EZR on R commander Version 1.55) or GraphPad Prism 9 for Windows (GraphPad Software, CA, USA).

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.